Calcitriol, the hormonally active form of vitamin D, has well known anti-cancer activity. We have recently identified four novel pathways of additional calcitriol action that inhibit breast cancer (BCa) growth. First, calcitriol directly inhibits aromatase expression in BCa cells. By reducing local estrogen synthesis, this action decreases the major growth stimulator of estrogen receptor (ER) positive BCa cells. Importantly, calcitriol exhibits tissue specific regulation of aromatase resulting in down-regulation in BCa and up-regulation in bone. This action would protect bone from the untoward side effect of osteoporosis due to estrogen deprivation routinely seen in aromatase inhibitor (AI) treated patients. Second, calcitriol inhibits prostaglandin (PG) synthesis by multiple actions including suppression of COX-2 expression. Since PGs are major stimulators of BCa growth, their inhibition adds to the effectiveness of calcitriol therapy. Third, PGs are major stimulators of aromatase in BCa and calcitriol's inhibition of PGs also indirectly reduces aromatase expression. Fourth, calcitriol directly down-regulates ER1 expression. Thus calcitriol acts by multiple pathways to attenuate BCa growth. We postulate that combination therapy of calcitriol with an AI will make AIs more effective and at lower doses thus increasing efficacy with a potential additional benefit of reduced osteoporosis. Objective: We propose to study these four pathways of calcitriol action and investigate the value of combination therapy of calcitriol with AIs to render the drugs more effective than either drug alone. These studies will rapidly pave the way to clinical trials of combination therapy in BCa patients. Specific Aims: Aim I will examine calcitriol regulation of aromatase and PG pathway genes and the effect on BCa cell growth. Aim II will study the mechanism for the differential regulation of aromatase. Aim III will examine the mechanism by which calcitriol down-regulates ER1, Aim IV will study calcitriol, AIs and their combinations in vivo in a xenograft mouse model. Study Design: Established BCa cell lines and primary BCa cells will be evaluated in culture for effects of calcitriol on aromatase and PG pathway genes. Calcitriol actions to inhibit these pathways will include measurement of both critical genes and down-stream biological responses. Mechanistic studies will address differential regulation of aromatase promoters in BCa and bone cells. The effects of calcitriol, and AIs will be assessed individually and in combination. All of these aspects will also be examined in an in vivo model of BCa xenografts in nude mice. Potential Benefits: The proposed studies will evaluate calcitriol, AIs and combinations for therapy of BCa patients. Calcitriol and AIs are approved drugs, orally active, safe and of modest cost. Progress to advance these combinations to clinical trials to treat and/or prevent BCa can be swift. We postulate that calcitriol plus an AI will show enhanced activity to prevent the growth and progression of BCa. We believe the data generated by this study will pave the way to support the rapid advancement of calcitriol-AI combination therapy to clinical trials for the treatment of BCa. This strategy will provide a safe and economical improvement to the usual therapeutic regimen administered to BCa patients. PROJECT NARRATIVE We postulate that calcitriol plus an AI will show enhanced activity to prevent the growth and progression of BCa. We believe the data generated by this study will pave the way to support the rapid advancement of calcitriol-AI combination therapy to clinical trials for the treatment of BCa. This strategy will provide a safe and economical improvement to the usual therapeutic regimen administered to BCa patients.